The NS3 helicase is an essential constituent of the replication machinery from Hepatitis C Virus (HCV), which is a positive-strand RNA virus that represents a major threat to public health. NS3 is a multifunctional molecular motor that hydrolyzes nucleotide triphosphates during the unwinding of both DNA and RNA duplexes. It is a member of helicase superfamily 2 (SF2), and it belongs to the DExH/D subgroup of enzymes that are involved in all aspects of RNA metabolism, including pre-mRNA splicing, RNA interference, translation, RNA degradation, and in many forms of viral replication. Despite the ubiquity of the DExH/D enzymes, their fundamental importance for viability of higher organisms, and their role in numerous human pathogens, there have been few studies on the molecular mechanisms for RNA unwinding and ribonucleoprotein remodeling by this family of proteins. Little is known about their nucleic acid specificity, their mode of translocation, the mechanism of strand displacement, or the coupling between ATP hydrolysis and work expended during unwinding. To address these issues, we propose to use NS3 as a model system for exploring the behavior of DExH/D proteins. It is an excellent prototype for numerous reasons: (a). NS3 has been structurally characterized and it is a phylogenetically typical of DExH/D member, (b). It is part of a large ribonucleoprotein machine that contains cofactors that modulate its behavior (like most DExH/D proteins), (c). It has a robust unwinding reaction that has been investigated qualitatively, (d). Information about NS3 is likely to be critical for the development HCV management strategies. We propose a comprehensive program for the biophysical characterization of NS3 helicase activity. We will examine behavior of the isolated NS3 enzyme and its properties in complex with modulatory cofactors NS4A and NS5B. DNA unwinding by NS3 will also be explored, as the reaction is mechanistically distinct and it may provide important insights into metabolic strategies of the virus. Experiments will be carried out by applying new pre-steady state kinetic methods that monitor the unwinding of combinatorial substrate libraries. This approach, together with coupled ATPase assays, studies on chemically modified duplex substrates, time-resolved footprinting and single molecule methods will allow us to characterize the microscopic behavior of NS3 in a variety of macromolecular contexts. The results will fundamentally extend our knowledge of helicase mechanism and facilitate the development of new HCV inhibitors.